Xylanase is an enzyme that randomly hydrolyzes β-1,4 bonds of xylan, which is a component of plant cell walls. The enzyme is expected to be used in a wide range of applications, such as a) saccharification of lignocellulosic raw materials, b) pulp bleaching, c) animal feed additives, d) detergent aids, and e) bread-making modifiers.
With regard to a) saccharification of lignocellulosic raw materials, a method for saccharification of a lignocellulosic raw material is known in which a monosaccharide serving as a fermentation substrate is produced from a lignocellulosic raw material using an enzyme. However, the expensiveness of enzymes such as cellulases and hemicellulases (xylanase or the like) that can be used for this saccharification method hinders practical use of this saccharification method. Addressing this problem, reutilization of enzymes used in the saccharification method has been proposed as a means effective for the reduction of the cost of the saccharification method (see, for example, Japanese Patent Application Laid-Open (JP-A) No. 2006-87319 (Patent Document 1), International Publication WO2011/065449 pamphlet (Patent Document 10) and WO2011/125056 pamphlet (Patent Document 11)).
Xylanase is an enzyme that breaks down hemicellulose (of which the main component is β-1,4-xylan), which is one of the main components of a lignocellulosic raw material. Therefore, xylanase is one of important enzymes in a method for saccharifying a lignocellulosic raw material. However, xylanase is known to have low stability.
Meanwhile, saccharification of a lignocellulosic raw material requires treatment in an acidic region of from pH 4.0 to pH 6.0 at a high temperature of from 40° C. to 60° C. for a few days. Thus, the low stability of xylanase is a hindrance to the reutilization of this enzyme.
A heat-resistant xylanase mutant derived from Trichoderma reesei (hereinafter abbreviated to “T. reesei”) (see, for example, WO 2007/115391 pamphlet (Patent Document 2) and WO 2007/115407 pamphlet (Patent Document 3)) exhibited a residual activity of 80% or higher even after heat treatment at from 50° C. to 70° C. for 30 minutes.
A Bacillus-derived heat-resistant xylanase (see, for example, JP-A No. 2004-121257 (Patent Document 4)) is also known to exhibit a residual activity of 90% or higher after heat treatment at 70° C. for 30 minutes.
In regard to b) pulp bleaching, it is known that the amount of bleaching agent to be used can be decreased by using xylanase in a pulp bleaching process.
In general, pulp bleaching in paper-making industry consists of a first stage which is a delignification treatment process (from pH 10 to 12, 80° C.) of removing lignin from pulp using an enzyme, and a second stage which is a bleaching process. The reason for performing the bleaching process in the second stage as described above is that about a few percent of lignin remains as a coloring component in the pulp even after the delignification treatment using an enzyme. Addition of a process of allowing xylanase to work, in addition to the delignification treatment process and the bleaching process, enables the breakage of hemicellulose chains bound to lignin and cellulose. As a result of this, lignin can be effectively removed, and it is expected that an effect in terms of decreasing the amount of bleaching agent to be used in the bleaching process can be obtained.
In order to efficiently perform the process of allowing xylanase to work, it is necessary to use a xylanase having properties such that the xylanase can tolerate treatment at about pH 10 and from 70° C. to 80° C. carried out for a few hours.
A heat-resistant xylanase mutant derived from T. reesei (see, for example, Patent Documents 2 and 3, and WO 2001/92487 (Patent Document 5) and WO 2003/046169 (Patent Document 6)) has an optimum reaction temperature of about 70° C. and an optimum reaction pH of from 7 to 8, demonstrating the possibility that the heat-resistant mutant xylanase can be used in a pulp bleaching process.
With regard to c) animal feed additives, animal feed is rich in plant fibers, and plant cell walls in the animal feed can be decomposed by adding xylanase. Therefore, the efficiency of absorption of plant nutrition by animals can be improved.
In cases in which animal feed is to be pelletized using xylanase, the xylanase is required to have stability with which the xylanase can tolerate treatment at from about 70° C. to about 90° C. for about 10 minutes. In addition, in order for the xylanase to work in the digestive organs of animals, the xylanase needs to exhibit high activity in an environment at about 40° C. and about pH 4.8.
Many of xylanases derived from filamentous fungi such as the genus Trichoderma and the genus Acremonium have an optimum pH of from 3 to 5 and an operable temperature range around 40° C.
The heat-resistant xylanase mutants described in Patent Document 2 Patent Document 3, WO 2001/27252 (Patent Document 7), and WO 2005/108565 (Patent Document 8) include mutants having an optimum pH of from about 5 to about 5.5.
d) Detergent Aid: The use of xylanase as a detergent aid can remove fluff on clothes.
Since recent drum-type washing machines are designed to save water, fine fluffing tends to occur as the number of times of washing increases. When the fluffing has occurred, re-soiling of clothes tends to occur.
The fluff can be removed by using xylanase as a detergent aid, and, therefore, re-soiling can also be prevented. Moreover, since the main components of stains attaching to clothes and derived from vegetables or fruits are cell walls which are derived from the vegetables or fruits and to which colorants are attached, effective washing can be carried out using xylanase in washing even in cases in which a water-saving-type drum-type washing machine is used.
In cases in which xylanase is to be used as a detergent aid, it is necessary to use a xylanase having alkali resistance and surfactant resistance. In addition, in cases in which xylanase is used in laundry cleaning, it is necessary to use a xylanase that stably works in a high temperature range of from 50° C. to 70° C.
A T. reesei-derived xylanase mutant having heat resistance and alkali resistance (for examples, see Patent Document 2, Patent Document 3, Patent Document 5, and Patent Document 6) has properties including an optimum temperature of from 62° C. to 75° C. and an optimum pH of from pH 7 to pH 8.
The xylanase mutant described in Patent Document 8 has an optimum pH of pH 5, which is at the acidic side. However, this mutant xylanase has an optimum temperature of 70° C., and maintains 100% activity at 60° C. and from pH 8 to pH 9 for at least 10 minutes.
Each of the heat-resistant and alkali-resistant xylanases derived from the genus Bacillus (see, for example, Patent Document 4 and JP-A No. 2007-54050 (Patent Document 9)) has properties including an optimum temperature range of from 50° C. to 70° C. and an optimum pH of from 7 to 8, and maintain 100%-activity at pH 9 and from 4° C. to 5° C. for a length of time of from 1 to 2 days.
In regard to e) bread-making modifiers, the quality of bread production can be improved by using xylanase as a bread-making modifier.
Xylanase has properties capable of decomposing the hemicellulose component of flour. Due to the decomposition of the hemicellulose component by xylanase, moisture bound to this component is released into dough, thereby changing the properties of the dough. As a result, the particle structure and the loaf volume of the produced bread are improved, leading to favorable quality preservation of the produced bread.
When making dough, large physical impact and pressure load are applied during a process of stirring and kneading ingredients, and a fermentation process requires a length of time of from 1 to 2 hours at a temperature of from 35° C. to 40° C.